Journal: bioRxiv
Article Title: RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion
doi: 10.1101/2025.09.23.677683
Figure Lengend Snippet: (A) MDA-MB-231 cells seeded on coverslips, surface labeled with MT1-MMP antibody at 4°C for 1 h Complete media was added, and cells were shifted to 37°C for 30 minutes to allow endocytosis. To remove surface-bound antibodies, an acid wash was given. After washing with PBS, serum-free media was added with or without HGF, and the cells were shifted to 37°C for 10 minutes to allow recycling from the endocytosed pool. Cells were fixed and immunostained for EEA1. The integral intensity of the MT1-MMP and the percentage of MT1-MMP antibody recycling were calculated. N = 3, n= 300; scale bars = 10 µm. The error bar represents means ± SD. Paired t-test, *** P< 0.001, Unpaired t-test, * P < 0.05. (B) MDA-MB-231 cells transfected with pHluorin MT1-MMP were seeded on gelatin-coated glass-bottom dishes. Time-lapse imaging was performed in the presence or absence of HGF stimulation using a Nikon TIRF microscope without any intervals for 300 frames. The flashes of pHluorin MT1-MMP, which represent exocytic events, were calculated using Motiontracking. scale bars = 10 µm. The error bar represents Means ± SEM. Unpaired t-test, ** P < 0.001. N = 4, n= 35. (C) MDA-MB-231 cells were seeded on gelatin-coated glass-bottom dishes and allowed to attach for 3h, followed by incubation with or without HGF for another 3h. Cells were fixed and immunostained for TKS5, MT1-MMP, and F-actin. Images were captured using a Nikon TIRF microscope. TKS5, MT1-MMP, and Actin-positive structures were identified using Motiontracking. The error bar represents Mean ±SEM, Unpaired t-test, * P < 0.01. scale bar: 10µm. N=3, n=120. (D) GFP-MT1-MMP expressing MDA-MB-231 cells were fixed and immunostained for MET. Images were captured using an LSM900 Airyscan super-resolution microscope. Images were processed using the Airyscan Joint deconvolution (jDCV) method. The image was converted to Maximum Intensity projection for representation. The inset shows a magnified view of the region indicated by the box. Scale bar: 10 µm, inset: 2 µm. (E) GFP or GFP-MT1-MMP expressing BT-549 cells were lysed and incubated with GST agarose beads bound to 25µg of GFP binding protein (GBP). Next washes were given to remove unbound proteins and proceeded for immunoblotting with GFP and MET. WCL: whole-cell lysates (F) MDA-MB-231 cells co-expressing GFP-MET and Cherry MT1-MMP were subjected to live-cell TIRF imaging. The inset represents an event at different time points. Scale bar: 10 µm, Inset: 2 µm. (G) BT-549 cells expressing MT1-MMP WT or a catalytic mutant alone or with V5-MET WT or M1250T mutant were seeded on gelatin-coated coverlips for 6h and fixed. Cells were immunostained for V5 and TKS5. Images were captured with the confocal microscope. The number of TKS5 puncta in the MT1-MMP and/or MET expressing cells was calculated and plotted. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 10µm. ** P<0.01, * P < 0.05, N=3, n=150
Article Snippet: MDA-MB-231 cells were trypsinized and counted, and 50,000 cells per well were seeded on the gelatin-coated coverslips for 24 h. After the cells had properly adhered and regained their morphology, antibody uptake was performed using anti-MT1-MMP antibody (R&D Systems, MAB9181-SP).
Techniques: Labeling, Transfection, Imaging, Microscopy, Incubation, Expressing, Super-Resolution Microscopy, Binding Assay, Western Blot, Mutagenesis
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